Understanding the importance of RNA binding proteins (RBPs)
RNA molecules serve a variety of essential roles in the cell. These include not only the well-characterized role as an information carrier between the DNA genome and translation at ribosomes but also non-coding roles in regulating gene expression, telomere maintenance, and a variety of other aspects of cellular physiology (1, 2). (Figure 1).
The processing of and regulation through RNA molecules is tightly controlled by RNA binding proteins (RBPs), which bind to RNAs through recognition of sequence and structural motifs and regulate RNA processing in cell-type, condition-specific, or temporal manners (3).
Recent studies have estimated over 1500 RBPs in the human genome, which play roles throughout the RNA life cycle (3). Mutation of proper RBP activity has been linked to cancer, Amyotrophic Lateral Sclerosis (ALS), and numerous other diseases, and the emergence of genome sequencing techniques will continue to rapidly expand our knowledge of RBPs causally mutated in disease (2).
Figure 1. Central dogma of molecular biology
1. Bandziulis RJ, Swanson MS, Dreyfuss G. RNA-binding proteins as developmental regulators. Genes & development.1989;3(4):431-7. PubMed PMID: 2470643.
2. Lukong KE, Chang KW, Khandjian EW, Richard S. RNA-binding proteins in human genetic disease. Trends in genetics
TIG. 2008;24(8):416-25. doi: 10.1016/j.tig.2008.05.004. PubMed PMID: 18597886.
3. Gerstberger S, Hafner M, Tuschl T. A census of human RNA-binding proteins. Nature reviews Genetics.
2014;15(12):829-45. doi: 10.1038/nrg3813. PubMed PMID: 25365966
4. Nussbacher JK, Batra R, Lagier-Tourenne C, Yeo GW. RNA-binding proteins in neurodegeneration:
Seq and you shall receive. Trends in neurosciences.2015;38(4):226-36. doi: 10.1016/j.tins.2015.02.003.
PubMed PMID: 25765321; PubMed Central PMCID: PMC4403644.
eCLIP has revolutionized the ability to identify RBP binding sites
Other non-CLIP methods have generally been limited to identification of transcript- (or large region-) level targets. Previous CLIP methods suffered from high experimental failure rates and technical challenges that hindered widespread adoption. In particular, the low efficiency of adapter ligation to RNA in previous CLIP approaches led to high PCR amplification requirements, which led to high PCR duplication rates and a low fraction of ‘usable reads’ (uniquely mapped reads that are not PCR duplicates).
To address these limitations, Eclipse Bioinnovations has optimized the eCLIP technology based on Van Nostrand, Yeo et al. Nature Methods 2016 paper to improve the efficiency of converting immunoprecipitated RNA into high-throughput sequencing libraries.
The eCLIP-seq method
Enhanced crosslinking and immunoprecipitation followed by high-throughput sequencing (eCLIP-seq) was developed to provide a robust and reproducible framework to identify RNA binding protein targets (Figure 2).
Figure 2. Schematic of the eCLIP methodology.
By altering the enzymatic steps performed to convert RNA into double-strand DNA libraries suitable for high-throughput sequencing, our eCLIP technology achieved a thousand-fold improvement in preparation efficiency. This innovation increased robustness in multiple ways:
-
- 3x decrease in unneeded sequencing costs.
- Increased signal-to-noise in identifying biologically-relevant binding sites.
- Reduce experimental failure rate, enabling rapid scaling to large-scale profiling
eCLIP Highlights
Figure 3. 102 eCLIP experiments show lower percentage of PCR duplications, relative to published iCLIP and other CLIP datasets
Increased efficiency, decreased PCR duplication
eCLIP-seq builds upon previous CLIP-seq methods to improve library preparation efficiency by nearly one thousand-fold, increasing experimental success rates and decreasing wasted sequencing due to PCR duplication (Figure 3).
Transcriptome-wide identification of RNA targets
eCLIP-seq identifies binding sites throughout RNAs, including binding to intronic and exonic positions and coding sequence and 3′ and 5′ untranslated regions, non-coding RNAs including lincRNAs and microRNAs, and retrotransposons and other RNA transcripts (Figure 4).
Figure 4. Example binding profiles for 5 RBPs.
Figure 5. Crosslink-termination analysis on RBFOX2
Single-nucleotide resolution
Reverse transcription often terminates at the protein-RNA crosslink site. By performing adapter ligation at the cDNA step, eCLIP can be used to identify binding sites and binding motifs with single-nucleotide resolution, depending on the target protein (Figure 5).
Mapping ADAR Binding Sites with eCLIP Technology
ADAR proteins play a pivotal role in mediating immune response to dsRNA and their
relevance in viral infection and cancer immunotherapy is becoming increasingly
recognized. ADAR1 and ADAR2 possess adenosine deaminase enzymatic activity which edits RNA by converting adenosine to inosine. Additionally, it has also been shown that ADAR binding alone can play a regulatory role, independent of its catalytic activity.
As a result, identifying ADAR-bound RNA targets irrespective of editing is also of significant importance. Eclipse’s eCLIP (Enhanced crosslinking and immunoprecipitation) technology allows identification of RNA-protein binding events with high accuracy, and it was recently utilized by the ENCODE consortium to profile binding sites of 150 RNA binding proteins. ADAR1 is the main editor of repetitive sites in cellular transcripts with p150 being an inducible isoform, that shuttles between nucleus and cytoplasm and p110 constitutively expressed with nuclear localization.
The ADAR eCLIP-seq Method
Schematic of the ADAR eCLIP methodology.
By altering the enzymatic steps performed to convert RNA into double-strand DNA libraries suitable for high-throughput sequencing, our eCLIP technology achieved a thousand-fold improvement in preparation efficiency. This innovation increased robustness in multiple ways:
-
- 3x decrease in unneeded sequencing costs.
- Increased signal-to-noise in identifying biologically-relevant binding sites.
- Reduce experimental failure rate, enabling rapid scaling to large-scale profiling
ADAR eCLIP Highlights
Peak Distribution of ADAR1
Genic distribution of p110 and p150 peaks. Both p110 and p150 peaks have strong distal intronic enrichment. p110 has 5’UTR and 3’UTR enrichment relative to CDS while p150 does not.
p110 Divergence pairs of Alu1 elements
ADAR1 p110 Binding of Alu1 Elements
~50% of all peaks in Alu1s are situated in paired (< 100 nt from each other) Alu1 elements in opposing orientations.
—> <— Convergent pairs
<— —>Divergent pairs (more common)
eCLIP Workflow
In eCLIP, RBP-RNA interactions are covalently linked using UV crosslinking of live cells. Cells are then lysed, and RNA is fragmented using limited RNase treatment. A specific RBP (and its bound RNA) is then immunoprecipitated using an antibody that specifically recognizes the targeted RBP. After ligation of a 3’ RNA adapter, immunoprecipitated material (as well as a paired input sample) are run on denaturing protein gels and transferred to nitrocellulose membranes.
A region from the protein size to 75 kDa above is cut from the membrane and treated with Proteinase K to release RNA. After cleanup, RNA is then reverse transcribed to ssDNA, after which a second adapter is ligated. PCR amplification is then used to obtain sufficient material for high-throughput sequencing (Figures 6, 7, 8)
Figure 6. eCLIP protocol overview.
eCLIP Data Analysis
Included in standard eCLIP services and eCLIP + Data Analysis kit
- HTML reports with figures related to significantly enriched peaks
- Fastq files of raw sequencing reads
- Adapter trimmed fastq files of reads with UMI and adapter sequences removed
- Bam files containing PCR-deduplicated read alignments to the genome
- Bigwig files which can be uploaded to a genome browser to view read density of sample
- Bed file of input-normalized peaks containing the location of each peak in the genome, along with the log2 fold change vs. input and p-value
Sample HTML Data Analysis Report
Contact us for details about additional analysis options.
Analyzing your own data
Standard analysis of eCLIP requires read adapter trimming, mapping to the appropriate genome, peak identification, and comparison against a paired input, knockout, or other suitable control sample (Figure 9). An example eCLIP analysis pipeline has been described by the ENCODE consortium here.
If you want to analyze your own data visit the Yeo lab pipeline on Github: github.com/YeoLab/eclip
Figure 9. eCLIP Data Analysis Pipeline. Eclipse Bioinnovations
eCLIP Experiment
What type of biological samples can be performed with eCLIP?
Most biological samples are amenable to eCLIP (cell lines, tissues and model organisms), with optimization required depending on endogenous RNase levels and other biological properties. Please contact us if you have a particular sample type of interest.
What do I need before starting an eCLIP experiment?
Antibody to immunoprecipitate the RNA binding protein of interest
Each eCLIP experiment uses an antibody to immunoprecipitate the RNA binding protein of interest from a biological sample.
Pre-validated antibodies are available for 150 RNA binding proteins
-Alternatively, we recommend antibody validation be performed prior to eCLIP using the eCLIP immunoprecipitation validation kit or anti-tag antibodies
UV Cross-linking
Biosamples should be UV cross-linked according to protocols available for:
– Tissues
Contact us for UV crosslinking protocols for Model Organisms
What are the sample requirements for an eCLIP experiment?
The quantity of starting material required for an eCLIP experiment depends significantly on the abundance of the protein you are studying. We would recommend however, starting with 200ug of RNA that has been quantified using our “RNA Fragmentation” guide at the protocol
What tags can I use for eCLIP?
We have in-house pre-validated antibodies for FLAG, V5, HA and MYC.
Do you usually recommend biological replicates, and if so, how many?
We recommend running at least duplicates for publication purposes, however triplicates always produce more reliable data.
Sequencing
Data Analysis
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